Research On The Regulation Of Osteogenesis And Adipogenesis Induced By BMP-9

Objective:1．To establish a stable and efficient method to regulate the expressionof target gene by AdEasy system and piggyBac system.2．To analyse the markers for osteogenesis and adipogenesis inducedby adenovirus vectors and/or stable cell line to confirm the function ofNell-1to osteogenesis and adipogenesis of mesenchymal stem cellsinduced by BMP-9.3．To regulate the expression of Creld2by adenovirus vectors toestablish the function of Creld2to BMP9-regulated terminal osteogenicdifferentiation of mesenchymal stem cells.Method:1．To construct the adenovirus vectors expressing Nell-1: amplify thefull sequence of Nell-1by Hi-Fi PCR and ligature the full sequence into theshuttle vectors, after recombination package adenovirus in HEK293cellsby AdEasy system and confirm it by fluorescence, RT PCR and otherfunction tests.2．To establish the stable cell line for overexpression the gene ofNell-1: ligature the full sequence of Nell-1into the vectors and increase the copy number of Nell-1in the genome of mesenchymal stem cells throughtransposition by piggyBac system, after siftting confirm the function by RTPCR and other function tests.3．To determine the role of Nell-1in BMP-9-induced osteogenesis andadipogenesin: analyse the markers for osteogenesis and adipogenesisinduced by adenovirus of BMP-9and/or Nell-1in mesenchymal stem cellsand the stable cell line for overexpression the gene of Nell-1by RT PCR,alkaline phosphatase activities, alizarin red staining, oil red O staining, cellcycle analysis and animal experiment.4．To determine the role of Creld2in osteogenesis induced by BMP-9:alter the expression of Creld2in mesenchymal stem cells by adenovirusand analysis alkaline phosphatase activities, alizarin red staining, stem cellimplantation and micro CT for osteogenesis induced by BMP-9.Results:1．Adenovirus vectors and stable cell line: adenovirus vectors caninfect mesenchymal stem cells and the fluorescence caused by theadenovirus vectors can be observed, the expression of target gene can beregulated by adenovirus and the stable cell line, confirmed by RT PCRanalysis on the total RNA from the stable cell line and mesenchymal stemcells infected by adenovirus.2．The regulation of Nell-1to the function of BMP-9:(1) In vitromesenchymal stem cells can not be induced terminal osteogenic differentiation by Nell-1alone effectively, but with BMP-9, the alkalinephosphatase activities and adipogenesis became weaker compare withBMP-9alone group but the mineralization confirmed by alizarin redstaining became stronger. When the expression of Nell-1was locked downby adenovirus, the adipogenesis induced by BMP-9became stronger.(2) Invivo Nell-1alone can not induce ectopic osteogenesis but with BMP-9thevolume of bone mass is smaller but the density is higher compare withBMP-9alone group. In histology, the bone mass from Nell-1plus BMP-9group is more mature and the scope of adipose tissue is smaller comparewith BMP-9alone group.3．The regulation of Creld2to osteogenesis induced by BMP-9:(1) Invitro all the makers for osteogenesis induced by BMP-9are in directproportion to the expression of Creld2.(2)Mesenchymal stem cellsimplantation assay reveals that exogenous Creld2augmentsBMP9-induced ectopic bone formation and mature mineralization, whereassilencing Creld2expression significantly diminishes BMP-9-induced boneformation and matrix mineralization.Conclusions:1．AdEasy system and piggyBac system can regulate the expression oftarget genes effectively.2．Nell-1can augment BMP-9-induced osteogenesis but inhibitsadipogenic differentiation of mesenchymal stem cells. 3．Creld2is an important mediator of BMP-9-regulated terminalosteogenic differentiation of mesenchymal stem cells.